Note: Descriptions are shown in the official language in which they were submitted.
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Description
Title of Invention: DRUG COMPRISING ARIPIPRAZOLE AND
CILOSTAZOL
Technical Field
[0001] The present invention relates to a medicament of aripiprazole and
cilostazol, and par-
ticularly relates to a combination of aripiprazole and cilostazol for use in
the treatment
and/or prevention of dementia, cognitive impairment, and/or vascular
depression.
Background Art
[0002] With society rapidly aging, increasing attention has been drawn to
dementia and
cognitive impairment. Diseases caused by vascular disorders, such as cerebral
stroke
and cerebral infarction, due to the aging of blood vessels have been becoming
more
serious problems, especially among the elderly. It is known that even if
adequate
treatment is given to a patient with a disease caused by a vascular disorder
and im-
provement is seen, the patient is likely to exhibit mental change or have
potential risk
of exhibiting mental change at a later stage. For example, vascular depression
known
to develop after treatment of vascular disorders is becoming an increasingly
serious
problem (see, for example, Patent Literature 1 and 2).
[0003] In the meantime, drugs having a high-level effect against platelet
aggregation are
known to be usable in the treatment and prevention of vascular disorders in
expectation
of their inhibitory effect on blood clots, such as cerebral infarction (see,
for example,
Patent Literature 2).
[0004] Aripiprazole, also expressed as
7- { 4- 14-(2,3-dichloropheny1)-1-piperazinyllbutoxy1-3,4-dihydro-2(1H)-
quinolinone, is
a carbostyril derivative and is known as an antipsychotic drug used in the
treatment of
mental disorders. Aripiprazole has also been studied for its applications in
treating de-
pression caused by vascular disorders (see, for example, Non-patent Literature
1).
Citation List
Patent Literature
[0005] PTL 1: Patent No. 4659693
PTL 2: JP2007-523121
Non-patent Literature
[0006] NPL 1: Y. R. Kim et al., Behavioural Brain Research, Vol.287, (2015)
pp.294-303.
Summary of Invention
Technical Problem
[0007] As noted above, diseases caused by vascular disorders are treated by
administering
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an antithrombotic drug or the like. However, further studies are needed to
elucidate the
effect of such antithrombotic drugs on mental disorders, and how the drugs
contribute
to improved treatment.
[0008] A main object of the present invention is to enable treatment and/or
prevention of
dementia, cognitive impairment, and vascular depression by using a combination
of
aripiprazole, known as an antipsychotic drug, with cilostazol, or the like.
Solution to Problem
[0009] The present inventors conducted extensive research to achieve the
above object and
found that a combination of aripiprazole with cilostazol (i.e., active
ingredients) can
produce a therapeutic and/or preventive effect on dementia, cognitive
impairment, and
vascular depression. The inventors further conducted research and completed
the
present invention.
[0010] The present invention was completed based on the findings.
[0011] Item 1. A combination comprising aripiprazole and cilostazol for use
in the treatment
and/or prevention of at least one member selected from the group consisting of
dementia, cognitive impairment, and vascular depression.
[0012] Item 2. The combination according to Item 1, which is a combination
drug
comprising the aripiprazole and the cilostazol.
[0013] Item 3. The combination according to Item 1, separately comprising
(A) a
medicament containing the aripiprazole and (B) a medicament containing the
cilostazol.
[0014] Item 4. The combination according to any one of Items 1 to 3,
wherein the dementia,
cognitive impairment, and vascular depression are caused by cerebral stroke.
[0015] Item 5. The combination according to any one of Items 1 to 4,
wherein the dementia
is at least one member selected from the group consisting of vascular dementia
and
senile dementia.
[0016] Item 6. The combination according to Item 5, wherein the vascular
dementia is cere-
brovascular dementia.
[0017] Item 7. The combination according to Item 5 or 6, wherein the
vascular dementia is
dementia induced after cerebral ischemia caused by cerebral stroke.
[0018] Item 8. The combination according to any one of Items 1 to 4,
wherein the cognitive
impairment is at least one member selected from the group consisting of
Alzheimer's
disease, learning disabilities caused by degenerative disorders, declines in
learning
ability, memory or cognitive dysfunction such as mild cognitive impairment,
senile
cognitive impairment, age-related cognitive decline, cerebral senility,
vascular
cognitive impairment, AIDS-associated dementia, electric-shock-induced
amnesia,
memory impairment associated with depression or anxiety, cognitive impairment
in
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Parkinson's disease, Down's syndrome, cerebral stroke, traumatic brain injury,
Huntington's disease, and attention deficit disorder.
[0019] Item 9. The combination according to Item 8, wherein the vascular
cognitive im-
pairment is cerebrovascular cognitive impairment.
[0020] Item 10. The combination according to Item 8 or 9, wherein the
vascular cognitive
impairment is cognitive impairment induced after cerebral ischemia caused by
cerebral
stroke.
[0021] Item 11. The combination according to any one of Items 1 to 4,
wherein the vascular
depression is cerebrovascular depression.
[0022] Item 12. The combination according to any one of Items 1 to 4, and
11, wherein the
vascular depression is depression induced by an age-related vascular disorder.
[0023] Item 13. The combination according to any one of Items 1 to 4, 11,
and 12, wherein
the vascular depression is depression induced after cerebral ischemia caused
by
cerebral stroke.
[0024] Item 14. The combination according to any one of Items 1 to 13,
which is ad-
ministered such that the daily dose of the aripiprazole is 0.01 to 300 mg/day.
[0025] Item 15. The combination according to any one of Items 1 to 14,
which is ad-
ministered such that the daily dose of the cilostazol is 1 to 300 mg/day.
[0026] Item 16. The combination according to any one of Items 1 to 15,
which is ad-
ministered such that the daily dose of the aripiprazole is 0.01 to 300 mg/day,
and the
daily dose of the cilostazol is 1 to 300 mg/day.
[0027] Item 17. The combination according to any one of Items 1 to 16,
wherein the
cilostazol is present in an amount of 0.1 to 100 parts by mass per part by
mass of the
aripiprazole in the combination, the combination being in the form of either a
com-
bination drug or medicament (A).
[0028] Item 18. A method for treating and/or preventing at least one member
selected from
the group consisting of dementia, cognitive impairment, and vascular
depression in a
patient in need of such a treatment and/or prevention, the method comprising
admin-
istering a combination of aripiprazole and cilostazol to the patient.
[0029] Item 19. The method according to Item 18, wherein the aripiprazole
and the
cilostazol are administered sequentially or simultaneously.
[0030] Item 20. The method according to Item 18 or 19, wherein the
dementia, cognitive im-
pairment, and vascular depression are caused by cerebral stroke.
[0031] Item 21. The method according to any one of Items 18 to 20, wherein
the dementia is
at least one member selected from the group consisting of vascular dementia
and senile
dementia.
[0032] Item 22. The method according to Item 21, wherein the vascular
dementia is cere-
brovascular dementia.
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[0033] Item 23. The method according to Item 21 or 22, wherein the vascular
dementia is
dementia induced after cerebral ischemia caused by cerebral stroke.
[0034] Item 24. The method according to any one of Items 18 to 20, wherein
the cognitive
impairment is at least one member selected from the group consisting of
Alzheimer's
disease, learning disabilities caused by degenerative disorders, declines in
learning
ability, memory or cognitive dysfunction such as mild cognitive impairment,
senile
cognitive impairment, age-related cognitive decline, cerebral senility,
vascular
cognitive impairment, AIDS-associated dementia, electric shock induced
amnesia,
memory impairment associated with depression or anxiety, cognitive impairment
in
Parkinson's disease, Down's syndrome, cerebral stroke, traumatic brain injury,
Huntington's disease, and attention deficit disorder.
[0035] Item 25. The method according to Item 24, wherein the vascular
cognitive im-
pairment is cerebrovascular cognitive impairment.
[0036] Item 26. The method according to Item 25, wherein the
cerebrovascular cognitive
impairment is cognitive impairment induced after cerebral ischemia caused by
cerebral
stroke.
[0037] Item 27. The method according to any one of Items 18 to 20, wherein
the vascular
depression is cerebrovascular depression.
[0038] Item 28. The method according to Item 18, 19, 20, or 27, wherein the
vascular de-
pression is depression induced by an age-related vascular disorder.
[0039] Item 29. The method according to Item 18, 19, 20, 27, or 28, wherein
the vascular
depression is depression induced after cerebral ischemia caused by cerebral
stroke.
[0040] Item 30. The method according to any one of Items 18 to 29, wherein
the daily dose
of the aripiprazole is 0.01 to 300 mg/day.
[0041] Item 31. The method according to any one of Items 18 to 30, wherein
the daily dose
of the cilostazol is 1 to 300 mg/day.
[0042] Item 32. The method according to any one of Items 18 to 31, wherein
the com-
bination is administered such that the daily dose of the aripiprazole is 0.01
to 300 mg/
day, and the daily dose of the cilostazol is 1 to 300 mg/day.
[0043] Item 33. A medicament comprising cilostazol for use in the treatment
and/or
prevention of at least one member selected from the group consisting of
dementia,
cognitive impairment, and vascular depression, wherein the cilostazol is
administered
to a patient being administered aripiprazole.
[0044] Item 34. A medicament comprising cilostazol for use in the treatment
and/or
prevention of at least one member selected from the group consisting of
dementia,
cognitive impairment, and vascular depression, wherein the cilostazol is
administered
simultaneously with aripiprazole to a patient in need of administration of
aripiprazole.
[0045] Item 35. A medicament comprising aripiprazole for use in the
treatment and/or
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prevention of at least one member selected from the group consisting of
dementia,
cognitive impairment, and vascular depression, wherein the aripiprazole is ad-
ministered to a patient being administered cilostazol.
[0046] Item 36. A medicament comprising aripiprazole for use in the
treatment and/or
prevention of at least one member selected from the group consisting of
dementia,
cognitive impairment, and vascular depression, wherein the aripiprazole is ad-
ministered simultaneously with cilostazol to a patient in need of
administration of
cilostazol.
[0047] Item 37. The medicament according to any one of Items 33 to 36,
wherein the
dementia, cognitive impairment, and vascular depression are caused by cerebral
stroke.
[0048] Item 38. The medicament according to any one of Items 33 to 36,
wherein the
dementia is at least one member selected from the group consisting of vascular
dementia, and senile dementia.
[0049] Item 39. The medicament according to Item 38, wherein the vascular
dementia is
cerebrovascular dementia.
[0050] Item 40. The medicament according to Item 38 or 39, wherein the
vascular dementia
is dementia induced after cerebral ischemia caused by cerebral stroke.
[0051] Item 41. The medicament according to any one of Items 33 to 37,
wherein the
cognitive impairment is at least one member selected from the group consisting
of
Alzheimer's disease, learning disabilities caused by degenerative disorders,
declines in
learning ability, memory or cognitive dysfunction such as mild cognitive
impairment,
senile cognitive impairment, age-related cognitive decline, cerebral senility,
vascular
cognitive impairment, AIDS-associated dementia, electric shock induced
amnesia,
memory impairment associated with depression or anxiety, cognitive impairment
in
Parkinson's disease, Down's syndrome, cerebral stroke, traumatic brain injury,
Huntington's disease, and attention deficit disorder.
[0052] Item 42. The medicament according to Item 41, wherein the vascular
cognitive im-
pairment is cerebrovascular cognitive impairment.
[0053] Item 43. The medicament according to Item 41 or 42, wherein the
vascular cognitive
impairment is cognitive impairment induced after cerebral ischemia caused by
cerebral
stroke.
[0054] Item 44. The medicament according to any one of Items 33 to 37,
wherein the
vascular depression is cerebrovascular depression.
[0055] Item 45. The medicament according to any one of Items 33 to 37, and
44, wherein
the vascular depression is depression induced by an age-related vascular
disorder.
[0056] Item 46. The medicament according to any one of Items 33 to 37, 44,
and 45,
wherein the vascular depression is depression induced after cerebral ischemia
caused
by cerebral stroke.
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[0057] Item 47. The medicament comprising cilostazol according to any one
of Items 33,
and 37 to 46, which is administered to a patient who is being administered
aripiprazole
in an amount of 0.01 to 300 mg/day.
[0058] Item 48. The medicament comprising cilostazol according to any one
of Items 34,
and 37 to 46, which is administered to a patient in need of administration of
arip-
iprazole in an amount of 0.01 to 300 mg/day.
[0059] Item 49. The medicament comprising cilostazol according to any one
of Items 33 to
46, which is administered such that the daily dose of the cilostazol is 1 to
300 mg/day.
[0060] Item 50. The medicament comprising aripiprazole according to any one
of Items 35,
and 38 to 46, which is administered to a patient who is being administered
cilostazol in
an amount of 1 to 300 mg/day.
[0061] Item 51. The medicament comprising aripiprazole according to any one
of Items 36
to 46, which is administered to a patient in need of administration of
cilostazol in an
amount of 1 to 300 mg/day.
[0062] Item 52. The medicament comprising aripiprazole according to any one
of Items 35
to 46, 50, and 51, which is administered such that the daily dose of the
aripiprazole is
0.01 to 300 mg/day.
[0063] Item 53. Use of a combination of aripiprazole and cilostazol in the
treatment and/or
prevention of at least one member selected from the group consisting of
dementia,
cognitive impairment, and vascular depression.
[0064] Item 54. The use according to Item 53, wherein the combination is a
combination
drug comprising the aripiprazole and the cilostazol.
[0065] Item 55. The use according to Item 53 or 54, wherein the dementia,
cognitive im-
pairment, and vascular depression are caused by cerebral stroke.
[0066] Item 56. The use according to Item 53, wherein the combination
separately
comprises (A) a medicament containing the aripiprazole and (B) a medicament
containing the cilostazol.
[0067] Item 57. The use according to any one of Items 53 to 56, wherein the
dementia is at
least one member selected from the group consisting of vascular dementia, and
senile
dementia.
[0068] Item 58. The use according to Item 57, wherein the vascular dementia
is cere-
brovascular dementia.
[0069] Item 59. The use according to Item 57 or 58, wherein the vascular
dementia is
dementia induced after cerebral ischemia caused by cerebral stroke.
[0070] Item 60. The use according to any one of Items 53 to 55, wherein the
cognitive im-
pairment is at least one member selected from the group consisting of
Alzheimer's
disease, learning disabilities caused by degenerative disorders, declines in
learning
ability, memory or cognitive dysfunction such as mild cognitive impairment,
senile
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cognitive impairment, age-related cognitive decline, cerebral senility,
vascular
cognitive impairment, AIDS-associated dementia, electric shock induced
amnesia,
memory impairment associated with depression or anxiety, cognitive impairment
in
Parkinson's disease, Down's syndrome, cerebral stroke, traumatic brain injury,
Huntington's disease, and attention deficit disorder.
[0071] Item 61. The use according to Item 60, wherein the vascular
cognitive impairment is
cerebrovascular cognitive impairment.
[0072] Item 62. The use according to Item 60 or 61, wherein the vascular
cognitive im-
pairment is cognitive impairment induced after cerebral ischemia caused by
cerebral
stroke.
[0073] Item 63. The use according to any one of Items 53 to 55, wherein the
vascular de-
pression is cerebrovascular depression.
[0074] Item 64. The use according to any one of Items 53 to 55, and 63,
wherein the
vascular depression is depression induced by an age-related vascular disorder.
[0075] Item 65. The use according to any one of Items 53 to 55, 63, and 64,
wherein the
vascular depression is depression induced after cerebral ischemia caused by
cerebral
stroke.
[0076] Item 66. The use according to any one of Items 53 to 65, wherein the
daily dose of
the aripiprazole is 0.01 to 300 mg/day.
[0077] Item 67. The use according to any one of Items 53 to 66, wherein the
daily dose of
the cilostazol is 1 to 300 mg/day.
[0078] Item 68. The use according to any one of Items 53 to 67, wherein the
daily dose of
the aripiprazole is 0.01 to 300 mg/day, and the daily dose of the cilostazol
is 1 to 300
mg/day.
[0079] Item 69. Aripiprazole and cilostazol for use in the treatment and/or
prevention of at
least one member selected from the group consisting of dementia, cognitive im-
pairment, and vascular depression.
[0080] Item 70. The aripiprazole and cilostazol according to Item 69, which
are ad-
ministered sequentially or simultaneously.
[0081] Item 71. The aripiprazole and cilostazol according to Item 69 or 70,
wherein the
dementia, cognitive impairment, and vascular depression are caused by cerebral
stroke.
[0082] Item 72. The aripiprazole and cilostazol according to any one of
Items 69 to 71,
wherein the dementia is at least one member selected from the group consisting
of
vascular dementia, and senile dementia.
[0083] Item 73. The aripiprazole and cilostazol according to Item 72,
wherein the vascular
dementia is cerebrovascular dementia.
[0084] Item 74. The aripiprazole and cilostazol according to Item 72 or 73,
wherein the
vascular dementia is dementia induced after cerebral ischemia caused by
cerebral
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stroke.
[0085] Item 75. The aripiprazole and cilostazol according to any one of
Items 69 to 71,
wherein the cognitive impairment is at least one member selected from the
group
consisting of Alzheimer's disease, learning disabilities caused by
degenerative
disorders, declines in learning ability, memory or cognitive dysfunction such
as mild
cognitive impairment, senile cognitive impairment, age-related cognitive
decline,
cerebral senility, vascular cognitive impairment, AIDS-associated dementia,
electric
shock induced amnesia, memory impairment associated with depression or
anxiety,
cognitive impairment in Parkinson's disease, Down's syndrome, cerebral stroke,
traumatic brain injury, Huntington's disease, and attention deficit disorder.
[0086] Item 76. The aripiprazole and cilostazol according to Item 75,
wherein the vascular
cognitive impairment is cerebrovascular cognitive impairment.
[0087] Item 77. The aripiprazole and cilostazol according to Item 75 or 76,
wherein the
vascular cognitive impairment is cognitive impairment induced after cerebral
ischemia
caused by cerebral stroke.
[0088] Item 78. The aripiprazole and cilostazol according to any one of
Items 69 to 71,
wherein the vascular depression is cerebrovascular depression.
[0089] Item 79. The aripiprazole and cilostazol according to any one of
Items 69 to 71, and
78, wherein the vascular depression is depression induced by an age-related
vascular
disorder.
[0090] Item 80. The aripiprazole and cilostazol according to Item any one
of Items 69 to 71,
78 and 79, wherein the vascular depression is depression induced after
cerebral
ischemia caused by cerebral stroke.
[0091] Item 81. The aripiprazole and cilostazol according to any one of
Items 69 to 80,
which are used such that the daily dose of the aripiprazole is 0.01 to 300
mg/day.
[0092] Item 82. The aripiprazole and cilostazol according to any one of
Items 69 to 81,
which are used such that the daily dose of the cilostazol is 1 to 300 mg/day.
[0093] Item 83. The aripiprazole and cilostazol according to any one of
Items 69 to 82,
which are used such that the daily dose of the aripiprazole is 0.01 to 300
mg/day and
the daily dose of the cilostazol is 1 to 300 mg/day.
[0094] Item 84. Use of aripiprazole and cilostazol for producing a
medicament for treating
and/or preventing at least one member selected from the group consisting of
dementia,
cognitive impairment, and vascular depression.
[0095] Item 85. The use according to Item 84, wherein the medicament is a
combination
drug comprising the aripiprazole and the cilostazol.
[0096] Item 86. The use according to Item 84 or 85, wherein the medicament
separately
comprises (A) a medicament containing the aripiprazole and (B) a medicament
containing the cilostazol.
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[0097] Item 87. The use according to any one of Items 84 to 86, wherein the
dementia,
cognitive impairment, and vascular depression are caused by cerebral stroke.
[0098] Item 88. The use according to any one of Items 84 to 87, wherein the
dementia is at
least one member selected from the group consisting of vascular dementia, and
senile
dementia.
[0099] Item 89. The use according to Item 88, wherein the vascular dementia
is cere-
brovascular dementia.
[0100] Item 90. The use according to Item 88 or 89, wherein the vascular
dementia is
dementia induced after cerebral ischemia caused by cerebral stroke.
[0101] Item 91. The use according to any one of Items 84 to 87, wherein the
cognitive im-
pairment is at least one member selected from the group consisting of
Alzheimer's
disease, learning disabilities caused by degenerative disorders, declines in
learning
ability, memory or cognitive dysfunction such as mild cognitive impairment,
senile
cognitive impairment, age-related cognitive decline, cerebral senility,
vascular
cognitive impairment, AIDS-associated dementia, electric shock induced
amnesia,
memory impairment associated with depression or anxiety, cognitive impairment
in
Parkinson's disease, Down's syndrome, cerebral stroke, traumatic brain injury,
Huntington's disease, and attention deficit disorder.
[0102] Item 92. The use according to Item 91, wherein the vascular
cognitive impairment is
cerebrovascular cognitive impairment.
[0103] Item 93. The use according to Item 91 or 92, wherein the vascular
cognitive im-
pairment is cognitive impairment induced after cerebral ischemia caused by
cerebral
stroke.
[0104] Item 94. The use according to any one of Items 84 to 87, wherein the
vascular de-
pression is cerebrovascular depression.
[0105] Item 95. The use according to any one of Items 84 to 87, and 94,
wherein the
vascular depression is depression induced by an age-related vascular disorder.
[0106] Item 96. The use according to any one of Items 84 to 87, 94, and 95,
wherein the
vascular depression is depression induced after cerebral ischemia caused by
cerebral
stroke.
[0107] Item 97. The use according to any one of Items 84 to 96, wherein the
daily dose of
the aripiprazole is 0.01 to 300 mg/day.
[0108] Item 98. The use according to any one of Items 84 to 97, wherein the
daily dose of
the cilostazol is 1 to 300 mg/day.
[0109] Item 99. The use according to any one of Items 84 to 98, wherein the
daily dose of
the aripiprazole is 0.01 to 300 mg/day and the daily dose of the cilostazol is
1 to 300
mg/day.
[0110] Item 100. A commercial package comprising a description concerning
the com-
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bination or the medicament according to the precedent Items,
wherein the description states that the combination or the medicament is
usable or to
be used in the treatment and/or prevention of at least one member selected
from the
group consisting of dementia, cognitive impairment, and vascular depression.
[0111] Item 101. A method comprising administering aripiprazole and
cilostazol to a patient
with at least one disease selected from the group consisting of dementia,
cognitive im-
pairment, and vascular depression,
wherein the aripiprazole and the cilostazol are administered as a single drug
or in-
dividual drugs,
wherein the aripiprazole and the cilostazol in the form of individual drugs
are ad-
ministered simultaneously or separately with a time interval.
Advantageous Effects of Invention
[0112] According to the present invention, aripiprazole, known as an
antipsychotic drug,
produces a remarkably excellent effect in the treatment and/or prevention of
dementia,
cognitive impairment, and vascular depression when used in combination with
cilostazol, having a blood clot inhibition activity, whose applications to
mental
disorders have not been fully known.
Brief Description of Drawings
[0113] [fig.11Fig. 1 shows the results of tests using middle cerebral artery
occlusion models.
[fig.21Fig. 2 shows the results of quantitative evaluation of CREB
phosphorylation in
mouse brain tissues.
[fig.31Fig. 3 shows the results of the measurement of Heme Oxygenase-1
expression
levels.
[fig.41Fig. 4 shows the results of a test using bilateral common carotid
artery stenosis
models.
Description of Embodiments
[0114] Aripiprazole, used in the present invention, is a carbostyril
derivative, also expressed
as
7- { 4- 14-(2,3-dichloropheny1)-1-piperazinyllbutoxy1-3,4-dihydro-2(1H)-
quinolinone.
Aripiprazole is also known as
7-[4-[4-(2,3-dichloropheny1)-1-piperazinyllbutoxy1-3,4-dihydro carbostyril,
Abilify,
OPC-14597, OPC-31, or BMS-337039. Aripiprazole has activity as an agonist for
the
serotonin receptor and dopamine receptor, and acts as an agonist or partial
agonist for
the serotonin 5HTIA receptor and the dopamine D2 receptor. Aripiprazole is a
dopamine-serotonin system stabilizer. The scope of the present invention
encompasses
the metabolites of aripiprazole. Dehydroaripiprazole is one of the metabolites
of arip-
iprazole.
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[0115] Cilostazol is also expressed as
6-[4-(1-cyclohexy1-1H-tetrazol-5-y1)butoxy1-3,4-dihydro carbostyril.
[0116] The diseases treated and/or prevented by the present invention is
dementia, cognitive
impairment, and vascular depression.
[0117] Examples of dementia, cognitive impairment, and vascular depression
include
disorders caused by cerebral stroke.
[0118] Examples of dementia include vascular dementia and senile dementia.
Vascular
dementia is induced particularly by a cerebrovascular disorder. Thus, vascular
dementia also includes frontotemporal dementia, alcoholic dementia, and
lacunar
dementia. More specific examples of vascular dementia include dementia induced
by a
cerebrovascular disorder, and examples of cerebrovascular disorders include
cerebral
stroke (e.g., cerebral infarction, cerebral hemorrhage, and subarachnoid
hemorrhage).
Still more specific examples of vascular dementia include dementia induced
after
cerebral ischemia caused by cerebral stroke.
[0119] Examples of cognitive impairment include Alzheimer's disease,
learning disabilities
caused by degenerative disorders, declines in learning ability, memory or
cognitive
dysfunction such as mild cognitive impairment, senile cognitive impairment,
age-
related cognitive decline, cerebral senility, vascular cognitive impairment,
AIDS-
associated dementia, electric shock induced amnesia, memory impairment
associated
with depression or anxiety, cognitive impairment in Parkinson's disease,
Down's
syndrome, cerebral stroke, traumatic brain injury, Huntington's disease, and
attention
deficit disorder. Examples of vascular cognitive impairment include
cerebrovascular
cognitive impairment, and examples of cerebrovascular cognitive impairment
include
cognitive impairment induced after cerebral ischemia caused by cerebral stroke
(for
example, cognitive impairment induced after cerebral ischemia caused by
cerebral in-
farction).
[0120] Vascular cognitive impairment is induced by a vascular disorder.
More specific
examples of vascular cognitive impairment include cognitive impairment induced
by a
cerebrovascular disorder. Examples of cerebrovascular disorders include
cerebral
stroke (e.g., cerebral infarction, cerebral hemorrhage, and subarachnoid
hemorrhage).
Still more specific examples of vascular cognitive impairment include
cognitive im-
pairment induced after cerebral ischemia caused by cerebral stroke(for
example,
cognitive impairment induced after cerebral ischemia caused by cerebral
infarction).
[0121] Vascular depression is induced after a vascular disorder. More
specific examples of
vascular dementia include depression induced by a cerebrovascular disorder.
Examples
of cerebrovascular disorders include cerebral stroke (e.g., cerebral
infarction, cerebral
hemorrhage, and subarachnoid hemorrhage). Examples of vascular depression
further
include depression induced by an age-related vascular disorder.
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[0122] Examples of vascular depression include post-cerebral stroke
depression, post-
cerebral infarction depression, and senile depression. For the diagnosis and
symptoms
of post-cerebral stroke depression and vascular depression, for example, DSM-5
(Diagnostic and Statistical Manual of Mental Disorders, 5th edition), Biol
Psychiatry
(1998; 43:705-712), etc., may be referred to.
[0123] The combination according to the present invention is useful as a
CREB (cAMP
response element binding protein) phosphorylation stimulating agent, a pCREB
(phosphated CREB) stimulating agent, or an HO-1 (heme oxygenase-1) stimulating
agent. The combination according to the present invention is also expected to
serve as
a BDNF (brain-derived neurotrophic factor) stimulating agent, a
G5K3/13(glycogen
synthase kinase 3 beta) phosphorylation stimulating agent, or a
pGSK3/13(phosphated
G5K3/13) stimulating agent.
[0124] The effect attributed to the fact that CREB is phosphorylated to
form phosphorylated
CREB (pCREB) is not particularly limited. For example, because of its ability
to
promote the synthesis of Bc1-2 (B-cell lymphoma 2), COX-2 (cyclooxygenase-2),
tyrosine hydroxylase, BDNF, which is a neurotrophic factor, and NGF (nerve
growth
factor), which is also a neurotrophic factor, pCREB is expected to bring
improvement
in learning ability or memory ability, anti-apoptotic action, increases in
neuro-
transmitter production, or enhancement of neurogenesis. The effect brought
about by
the production of pCREB also includes the effect disclosed in the literature
Carlos A.
Saura (Rev. Neurosci., Vol. 22(2): 153-169, 2011). HO-1 is known as a
cytoprotective
protein that protects cells from damage caused by oxidative stress. Thus, HO-1
is also
expected to have, for example, the effect disclosed in the following
literature: Stephan
W. Ryter, Physiol Rev 86: 583-650, 2006. In addition, given the promoting
effect on
the production of BDNF or phosphorylation of GSK3/13, HO-1 shows its promising
ap-
plications in Alzheimer's dementia, depression, anxiety disorder, bipolar
disorder,
schizophrenia, autism spectrum disorder (developmental disorder), Parkinson's
disease,
tauopathy (tau protein abnormality), or other diseases.
[0125] The following describe an embodiment of the present invention in
detail.
[0126] The combination according to the present invention comprises, as
active ingredients,
aripiprazole and cilostazol. The combination, for example, may be in the form
of a
combination drug comprising aripiprazole and cilostazol in a single
medicament, or in
the form of a combination separately comprising (A) a medicament containing
arip-
iprazole and (B) a medicament containing cilostazol.
[0127] The combination is used in the form of a typical pharmaceutical
preparation. Pharma-
ceutical preparations are prepared using diluents and excipients generally
used in phar-
maceuticals, such as fillers, extenders, binders, humectants, disintegrators,
surfactants,
and lubricants. For these preparations, various pharmaceutical forms can be
selected in
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accordance with the therapeutic purposes, and typical examples include
tablets, pills,
powders, fluids, suspensions, emulsions, granules, capsules, suppositories and
in-
jectable drugs (e.g., fluids and suspensions).
[0128] When the combination comprises medicament (A) and medicament (B),
the pharma-
ceutical forms of medicament (A) and medicament (B) are not particularly
limited, and
may be the same or different, as long as the medicaments are in the form
listed above.
[0129] When the combination is formed into tablets, a wide variety of
carriers conven-
tionally known in this technical field can be used. Examples of such carriers
include
excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch,
calcium
carbonate, kaolin, crystalline cellulose, and silicic acid; binders such as
water, ethanol,
propanol, simple syrup, glucose solution, starch solution, gelatin solution,
car-
boxymethyl cellulose, shellac, methylcellulose, potassium phosphate, and
polyvinylpyrrolidones; disintegrators such as dry starch, sodium alginate,
agar powder,
laminaran powder, sodium hydrogen carbonate, calcium carbonate,
polyoxyethylene
sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride,
starch, and
lactose; disintegration inhibitors such as sucrose, stearin, cacao butter, and
hy-
drogenated oils; absorption enhancers such as quaternary ammonium base and
sodium
lauryl sulfate; humectants such as glycerol and starch; adsorbents such as
starch,
lactose, kaolin, bentonite, and colloidal silica; and lubricants such as
purified talc,
stearate, boric acid powder, and polyethylene glycol. Tablets can also be
formed as
tablets with ordinary coatings, if necessary. Examples of such tablets include
sugar-
coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated
tablets,
double-layer tablets, and multilayer tablets.
[0130] When the combination is formed into pills, a wide variety of
carriers conventionally
known in this technical field can be used. Examples of usable carriers include
ex-
cipients such as glucose, lactose, starch, cacao butter, hydrogenated
vegetable oil,
kaolin, and talc; binders such as powdered gum arabic, powdered tragacanth,
gelatin,
and ethanol; and disintegrators such as laminaran and agar.
[0131] When the combination is formed into suppositories, a wide variety of
carriers con-
ventionally known in this technical field can be used. Examples include
polyethylene
glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, and
semisynthetic glyceride.
[0132] Capsules are prepared according to an ordinary method typically by
mixing an active
ingredient compound with one or more of various carriers as listed above, and
packing
the mixture in hard gelatin capsules, soft capsules, or the like.
[0133] When the combination is prepared as an injectable drug, it is
preferable that the fluid,
emulsion, or suspension is sterilized, and is isotonic with blood. When the
combination
is formed into a fluid, emulsion, or suspension, any of diluents
conventionally used in
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this technical field can be used. Examples of usable diluents include water,
alcohols,
such as methanol, ethanol, propanol, isopropanol, ethoxylated isostearyl
alcohols, and
polyoxylated isostearyl alcohols; macrogols; propylene glycols;
polyoxyethylene
sorbitan fatty acid esters; ketones, such as acetone; ethers, such as
tetrahydrofuran; and
dimethylformamide.
[0134] When prepared in the form of an injectable drug, the combination may
contain salt,
glucose, or glycerin in such a sufficient amount as to form an isotonic
solution. A
typical solubilizing agent, a buffer, or a soothing agent may be added. The
combination
may also optionally contain a coloring agent, a preserving agent, a flavoring,
a
sweetening agent as well as one or more other drugs.
[0135] When the combination is a combination drug, the aripiprazole content
is not par-
ticularly limited, and can be suitably selected from a wide range. However,
arip-
iprazole is preferably present in an amount of about 0.1 to 35% by mass in the
com-
bination drug. The cilostazol content is not particularly limited, and can
suitably
selected from a wide range. However, cilostazol is preferably present in an
amount of
about 0.1 to 35% by mass in the combination drug.
[0136] When the combination separately comprises medicament (A) and
medicament (B),
the aripiprazole content is not particularly limited, and can be suitably
selected from a
wide range. However, aripiprazole is typically present in an amount of about
0.1 to
70% by mass in medicament (A). The cilostazol content is not particularly
limited, and
can be suitably selected from a wide range. However, cilostazol is typically
present in
an amount of about 0.1 to 70% by mass in medicament (B).
[0137] The cilostazol content in the combination (i.e., a combination drug
or medicament
(B)) is preferably about 0.1 to 100 parts by mass per part by mass of
aripiprazole
present in the combination (i.e., a combination drug or medicament (A)).
[0138] The method for administering the combination is not particularly
limited, and the
combination is administered in accordance with the pharmaceutical form, the
patient's
age, the gender and other conditions, the extent of the disease, or the like.
For example,
the combination is administered orally, when in the form of tablets, pills,
fluids, sus-
pensions, emulsions, granules, or capsules. When in the form of an injectable
drug, the
combination is intravenously administered as a single drug or as a mixture
containing a
typical replacement fluid such as glucose and amino acids. The combination in
the
form of injectable drug is optionally administered as a single drug through an
intra-
muscular, intracutaneous, subcutaneous, or intraperitoneal route. The
combination is
administered rectally when in the form of a suppository.
[0139] When the combination comprises medicament (A) and medicament (B),
medicament
(A) and medicament (B) may be administered sequentially or simultaneously.
[0140] When medicament (A) and medicament (B) are administered
sequentially,
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medicament (A) may be administered first, and medicament (B) next, or vice
versa.
Specific examples include the method comprising administering cilostazol using
medicament (B) to a patient already being administered aripiprazole using
medicament
(A) and the method comprising administering aripiprazole using medicament (A)
to a
patient already being administered cilostazol using medicament (B). The term
"patient" as used herein include patients with diseases and/or disorders
described
above.
[0141] As used herein, "being administered aripiprazole" and "being
administered
cilostazol", respectively, refer to the state before aripiprazole is
substantially cleared
from blood, and the state before cilostazol is substantially cleared from
blood.
[0142] Specific examples of simultaneous administration of medicament (A)
and
medicament (B) includes a method comprising simultaneously administering (A) a
medicament containing aripiprazole and (B) a medicament containing cilostazol
to a
patient in need of administration of aripiprazole using medicament (A) or a
patient in
need of administration of cilostazol using medicament (B). The patient in need
of ad-
ministration of aripiprazole or cilostazol is a patient with diseases and/or
disorders
described above.
[0143] The dose of the combination is suitably selected in accordance with
the dose
regimen, the patient's age, the gender and other conditions, the extent of the
disease, or
the like. However, the daily dose of aripiprazole is typically about 0.01 to
300 mg/day,
preferably 0.1 to 100 mg/day, and more preferably 1 to 30 mg/day. The daily
dose of
cilostazol is typically about 1 to 300 mg/day, preferably 10 to 250 mg/day,
and more
preferably 50 to 200 mg/day.
[0144] Examples
The following Examples and Comparative Examples describe the present invention
in more detail. However, the present invention is not limited to the following
em-
bodiments.
[0145] Example 1
1.1: In Vivo Test (Middle Cerebral Artery Occlusion Model)
In this experiment, 30 male 10-week-old C57BL/6J mice were divided into the
following groups. To each group, 6 mice were allocated.
[0146] Grouping
1) Control Group
2) Group Administered Vehicle
3) Group Administered Cilostazol Alone
4) Group Administered Aripiprazole Alone
5) Group Administered Cilostazol and Aripiprazole
A 20% DMSO aqueous solution served as a vehicle. The dose of cilostazol and
the
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dose of aripiprazole were each 3 mg/kg. The dose of cilostazol and the dose of
arip-
iprazole for the group administered cilostazol and aripiprazole were also 3
mg/kg each.
The medicaments were each dissolved in a 20% DMSO aqueous solution and ad-
ministered to the mice.
[0147] Test Schedule
All of the mice were given a 14-day habituation period. On the day following
the
final day of the habituation period, the 4 mouse groups other than the control
group
underwent surgery, described later, to induce middle cerebral artery
occlusion. From
day 2 after the surgery, the mice of each group were orally administered their
medicament once a day over 16 consecutive days as determined for each group
described above and also given chronic mild stress described later. At the
points 4
weeks, 5 weeks and 6 weeks after the middle cerebral artery occlusion surgery,
all of
the mice were subjected to the open field test, sucrose preference test
(sucrose con-
sumption test), and forced swimming test, which are described later. At the
point 6
weeks after the middle cerebral artery occlusion surgery, all of the mice were
subjected
to the Morris water maze test. After the completion of the Morris water maze
test, the
brains of all of the mice were collected to prepare immunostaining samples of
the brain
tissues, and the state of the brain tissues were evaluated.
[0148] 1.2: Middle Cerebral Artery Occlusion Surgery
Male C57BL/6J mice underwent surgery to induce middle cerebral artery
occlusion
("MCAO") under anesthesia with 1.5% isoflurane (in 80% N20 and 20% 02). The
depth of anesthesia was checked by the absence of cardiovascular changes in
response
to tail pinch. The rectal temperature was kept at 36.5-37.5 C using a
thermostatically
controlled heating mat (Panlab, Harvard Apparatus) during the surgery. A
silicone-
coated monofilament was introduced into the internal carotid artery and
allowed to
advance so as to occlude the internal carotid artery. The monofilament was
withdrawn
from the internal carotid artery 30 minutes after occlusion. The cerebral
blood flow of
all of the mice subjected to the surgery was measured to confirm the
achievement of
consistent and similar levels of ischemic induction. To measure the cerebral
blood
flow, a laser Doppler (PeriFlux Laser Doppler System 5000; Perimed, Stockholm,
Sweden) with a flexible probe was used, and the obtained data were
continuously
recorded using a data acquisition and analysis system (PowerLab, AD
Instruments,
Medford, MA) and stored in a computer.
[0149] 1.3: Chronic Mild Stress (CMS)
Chronic mild stress was caused by randomly adding the following 7 different
stresses
for 16 consecutive days.
1) Food and water deprivation for 20 hours
2) Water deprivation for 18 hours
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3) 45 cage tilt for 17 hours
4) Overnight illumination (placed under illumination for a total of 36
consecutive
hours)
5) Soiled cage (200 ml of water was added to 100 g of sawdust, and cages were
bedded
with the water-containing sawdust; the mice were placed in the cages for 21
hours)
6) Forced swimming in 4 C water (5 minutes)
7) Pairs of mice were each placed in one cage (bottom surface: 28 cm x 42 cm,
height:
20 cm) for 2 hours.
[0150] 1.4: Open Field Test
The mice were placed at the center of cages made from white polyethylene
(30-cm-square, height: 40 cm). The distance over which the mice traveled in a
prede-
termined time was monitored with a video-tracking system using the Smart
software
(Panlab, Barcelona, Spain).
[0151] 1.5: Sucrose Preference Test
After food and water deprivation for 20 hours, a sucrose preference test was
performed. Two bottles, one containing a 1% sucrose aqueous solution and the
other
containing water, were set in each cage, and the mice were placed in the cages
for 1
hour, followed by measurement of the amount drank by the mice for both
bottles. At
the point 30 minutes after the start of the 1-hour test, the two bottles were
weighed, and
the position of the two bottles was changed, followed by continuation of the
test for the
remaining 30 minutes. The baseline value for the sucrose preference test was
de-
termined by measuring the value before starting to add the chronic mild
stress. The
sucrose preference was calculated according to the following equation:
sucrose aqueous solution intake (g) + water intake (g).
[0152] 1.6: Forced Swim Test
Antidepressant-like activities were evaluated by performing the forced swim
test.
One day before the test, mice were forced to swim in a glass cylinder (height:
15 cm,
diameter: 10 cm) containing 25 C water for 15 minutes. On the test day, the
mice were
forced to swim in the water-containing glass cylinder for 5 minutes in the
same
manner, and the behaviors of the mice were recorded with a digital camera
(E8400,
Nikon Corporation, Japan), and the immobile time was measured.
[0153] 1.7: Morris Water Maze Test
Spatial learning and memory decline were evaluated by performing the Morris
water
maze test. The Morris water maze test was performed in accordance with the
procedure
of Takeda S. et al. (Brain Res., (2009), Vol.1280, 137-147). The maze
consisted of a
1.15-m-diameter circular pool painted flat white, and was provided with a
10-cm-diameter circular platform halfway between the center of the pool and
the edge.
The platform was set 1 cm below the surface of the water. The water
temperature of
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the circular pool was maintained at 19-21 C. The circular pool was located in
a test
room that contains many cues external to the maze. The position of the cues
remained
unchanged throughout the water maze test. Between a series of test trials, the
mice
were randomly placed in one of four directional starting locations (north,
south, east,
and west) in the pool facing the wall of the circular pool, and the test was
started. The
mice were given a maximum of 180 seconds to reach the platform submerged in
water.
When a mouse was unable to reach the platform within 180 seconds, the mouse
was
guided to the platform and placed on the platform for 10 seconds. Swimming was
video-recorded, and the escape latency to reach the platform was analyzed
using the
Smart software (Panlab, Barcelona, Spain). The mean of latency time from the
platform was analyzed.
[0154] 1.8: Immunofluorescence
Mice received intracardial perfusion with PBS followed by fixative containing
4%
paraformaldehyde under chloral hydrate anesthesia. Thereafter, the brains were
removed from the mice and fixed by immersion in 4% paraformaldehyde for 24
hours.
The fixed brains were immersed in a 30% sucrose solution for 48 hours at 4 C.
The
obtained brains were frozen and 30-1tm-thick sections were obtained with a
cryostat.
The obtained sections were incubated overnight with the following primary
antibodies
in an antibody dilution buffer (PBS containing 1% of BSA and 0.3% of triton X-
100)
at 4 C: the primary antibodies are phospho-CREB (pCREB, 5er133, Santa Cruz,
CA,
USA), neuronal nuclei (NeuN, Millipore Corporation), and tyrosine hydroxylase
(TH,
Millipore Corporation). After being washed with PBS, the sections were
incubated
with a fluorescent secondary antibody (Vector Laboratories, Inc., Burlingame,
CA,
USA) and DAPI (Invitrogen Corporation, Carlsbad, CA, USA) for 2 hours and 30
minutes in a darkroom. Images were captured with a fluorescence microscope
(Carl
Zeiss, Inc., Goettingen, Germany).
[0155] 1.9: Statistical Analysis
All of the data were expressed as the mean standard error of the mean. The
sta-
tistical analysis was performed using ANOVA. When a statistically significant
effect is
found, post hoc analysis is performed to detect the difference between the
groups. A
value of P<0.05 was accepted as being statistically significant.
[0156] 1.10: Test Results
1.10.1
Fig. 1 shows the results of the open field test, sucrose preference test,
forced
swimming test, and Morris water maze test. In Fig. 1, graphs A, B, C, and D re-
spectively show the results of the open field test, the results of the sucrose
preference
test, the results of the forced swimming test, and the results of the Morris
water maze
test. #13<0.05, "P<0.01, and "413<0.001 versus the control group. P<0.05,
'13<0.01, and
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'13<0.001 versus the group administered vehicle. sP<0.05, $$P<0.01, and
$$$P<0.001
versus the group administered cilostazol alone. 843<0.05, 8L8L13<0.01, and
"L.843<0.001
versus the group administered aripiprazole alone.
[0157] In the open field test, the total distance traveled by the mice in
the group ad-
ministered vehicle was significantly shorter than that of the mice in the
control group.
However, the total distance traveled by the mice in the group administered
cilostazol
and aripiprazole was significantly longer than that of the mice in the group
ad-
ministered vehicle.
In the sucrose preference test, the mice in the group administered cilostazol
and arip-
iprazole exhibited a significantly higher intake of the sucrose aqueous
solution than the
mice in the group administered vehicle, the mice in the group administered
cilostazol
alone, or the mice in the group administered aripiprazole alone.
In the forced swimming test, the mice in the group administered vehicle
exhibited a
significantly shortened immobile time than the mice in the control group.
However, the
mice in the group administered cilostazol and aripiprazole exhibited a
significantly
extended immobile time than the mice in the group administered vehicle.
In the Morris water maze test, the mice in the group administered vehicle
exhibited
significantly longer escape latency than the mice in the control group.
However, the
mice in the group administered cilostazol and aripiprazole exhibited
significantly
shorter escape latency than the mice in the group administered vehicle, the
mice in the
group administered cilostazol alone, or the mice in the group administered
aripiprazole
alone.
[0158] 1.10.2
Fig. 2 shows the results of quantified immunostaining analysis. The vertical
axis
indicates the sum of the number of cells positive for both pCREB and NeuN and
the
number of cells positive for both pCREB and TH. In Fig. 2, "P<0.01 and
###P<0.001
versus the control group, 13<0.05, 'P<0.01, and 'P<0.001 versus the group ad-
ministered vehicle, $$P<0.01 and $$$P<0.001 versus the group administered
cilostazol
alone, and 843<0.05, 8L8L13<0.01, and "L.843<0.001 versus the group
administered arip-
iprazole alone.
[0159] The mice in the group administered vehicle exhibited a significantly
lower sum of
the number of cells positive for both pCREB and NeuN and the number of cells
positive for both pCREB and TH in the brain tissues of any of the striatum,
hip-
pocampus, or midbrain than the mice in the control group. However, the mice in
the
group administered cilostazol and aripiprazole exhibited a significantly
higher sum of
the number of cells positive for both pCREB and NeuN and the number of cells
positive for both pCREB and TH in the brain tissues than the mice in the group
ad-
ministered vehicle, the mice in the group administered cilostazol alone, or
the mice in
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the group administered aripiprazole alone.
[0160] Example 2
2.1: In Vitro Test
The effect of aripiprazole and/or cilostazol on neuronal cells was evaluated.
Specifically, the expression level of heme oxygenase-1 ("H0-1") was measured
using
Western blotting. HO-1 is a neuroprotective protein that protects cells from
damage
caused by oxidative stress.
HT22 neuronal cells (mouse hippocampus-derived neuronal cell line) were
cultured
in a Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine
serum, 100 units/mL of penicillin, and 100 [ig/mL of streptomycin. Thereafter,
the
cultured cells were divided into the following 4 groups: [1] a non-treated
group, [2] a
group treated with aripiprazole (1 [1M), [3] a group treated with cilostazol
(1 [1M), and
[4] a group treated with aripiprazole (1 [1M) + cilostazol (1 [1M), and the
divided cells
were subjected to respective treatments. Each treatment was performed by
exposing
the cell groups to respective substances for 6 hours. Thereafter, the treated
cells were
collected and lysed in a lysis buffer to prepare samples for electrophoresis,
followed by
polyacrylamide gel electrophoresis. The proteins separated by polyacrylamide
gel elec-
trophoresis were transferred onto PVDF membranes, and the PVDF membranes were
treated with blocking buffer. The PVDF membranes were then sequentially
treated
with the primary antibody of HO-1 (Assay Design), the secondary antibody, and
a
chemiluminescent reagent in the Supersignal West Dura Extended Duration
Substrate
Kit (Pierce Chemical). Subsequently, the signals from bands on the PVDF
membranes
were measured with a detector. The expression level of HO-1 protein was
normalized
to 13-actin level for evaluation.
[0161] 2.2: Statistical Analysis
The results were expressed as the mean standard error of the mean. The
statistical
analysis was performed using ANOVA. In the confirmation of statistically
significant
differences, Bonferroni's multiple comparison test was performed as a post hoc
analysis to detect the differences between groups.
[0162] 2.3: Test Results
Fig. 3 shows the results of measurement of the HO-1 protein expression levels.
In
Fig. 3, **13 < 0.01 versus the non-treated sample, IP <0.05 versus the sample
treated
with aripiprazole (ARP 1 [1M), and sP < 0.05 versus the sample treated with
cilostazol
(CSZ 1 [1M).
[0163] The HT22 neuronal cells treated with both aripiprazole and
cilostazol exhibited a sig-
nificantly higher HO-1 expression level than the non-treated HT22 neuronal
cells, the
HT22 neuronal cells treated with aripiprazole alone, or the HT22 neuronal
cells treated
with cilostazol alone.
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[0164] Example 3
3.1: In Vivo Test (Bilateral Common Carotid Artery Stenosis Model)
Mouse models with bilateral common carotid artery stenosis were prepared, and
the
Morris water maze test was performed on the mice models to evaluate the
spatial
learning and memory abilities. Forty male 10-week-old C57BL/6J mice (purchased
from Koatech, Seoul, Korea) were divided into the following groups for use. To
each
group, 8 mice were allocated.
[0165] Grouping
1) Control Group
2) Group Administered Vehicle
3) Group Administered Aripiprazole Alone
4) Group Administered Cilostazol Alone
5) Group Administered Cilostazol and Aripiprazole
A 25% DMSO aqueous solution served as a vehicle. For the group administered
cilostazol and aripiprazole as well, the dose of aripiprazole was 0.5 mg/kg
and the dose
of cilostazol was 20 mg/kg. Each medicament was dissolved in a 25% DMSO
aqueous
solution and orally administered to the mice.
[0166] Test Schedule
All of the mice were given a 5-day habituation period. On the day following
the final
day of the habituation period, the mice of each group underwent surgery to
induce
stenosis in the bilateral common carotid arteries. From day 2 to day 29 after
surgery,
the mice of each group were orally administered their medicament as determined
for
each group described above. The Morris water maze test was performed 3 weeks
after
the stenosis surgery.
[0167] 3.2: Bilateral Common Carotid Artery Stenosis Surgery
The mice underwent surgery to induce bilateral common carotid artery stenosis
("BCCAS") in accordance with the procedure disclosed in Shibata M. et al.
(Stroke,
(2004), Vol.35, 2598-2603). A midline neck incision was made to the mice to
expose
the bilateral common carotid arteries, and the arteries were separated from
the sur-
rounding tissues. A microcoil with an inner diameter of 0.18 mm was attached
to the
outer side of the separated bilateral common carotid arteries. The control
group also
underwent the same BCCAS surgery except that a microcoil was not attached.
[0168] 3.3: A spatial and memory ability test
To the purpose for assessment of the above, the Morris water maze test was
performed in accordance with the procedure disclosed in Park SH. et al.
(Biochem.
Biophys. Res. Com., (2011), Vol.408, 602-608). The maze consisted of a
1.15-m-diameter circular pool painted flat white, and a sufficient amount of
water
filled the circular pool. Of the 4 quadrants constituting the circular pool,
one quadrant
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was provided with a 10-cm-diameter circular platform. The platform was set 1
cm
below the surface of the water in the circular pool. To make the platform
invisible
under the water surface, powdered milk was dissolved in the water filling the
circular
pool. The water temperature was maintained at 19-21 C.
In the preliminary training, the time a mouse spent from finding the platform
until
standing up on the platform with four limbs touched on the platform was
determined to
be the escape latency. The training continued for 5 consecutive days, during
which the
platform remained in the same location. The mice were given a maximum of 90
seconds to reach the platform submerged in water. When a mouse was unable to
reach
the platform within 90 seconds, the mouse was guided to the platform, where
the
mouse stayed for 10 seconds, and was returned to the cage. In this case, the
escape
latency was recorded as 90 seconds.
Before BCCAS surgery, the mice in each group were subjected to training as
listed
below.
[0169] Day 1: the mice were allowed to swim freely for 90 seconds in the
circular pool
without the platform being set.
Day 2: the platform was set inside the circular pool, and the mice were
trained to find
the platform one time.
Day 3: the mice were trained to find the platform twice.
Day 4: the mice were trained to find the platform three times.
[0170] A spatial and memory ability test was performed 3 weeks after the
BCCAS surgery.
The mice were allowed to swim freely in the circular pool with no platform.
The
swimming was video-recoded, and analyzed with the Smart software (Panlab,
Barcelona, Spain). The ratio (%) of the time during which the mice spent
within the
quadrant where the platform was placed to the time during which the mice
stayed in
the circular pool in the training was calculated.
[0171] 3.4: Statistical Analysis
All of the data were expressed as the mean standard error of the mean.
Student's t-
test was used to determine significant differences. The effects of
Aripiprazole alone,
Cilostazol alone and their combination on the escape latency and on the time
spent in
the target quadrant in the Morris water maze test were analyzed with repeated
ANOVA
followed by the post hog Bonferroni's multiple comparison tests. A value of
P<0.05
was accepted as being statistically significant.
[0172] 3.5: Test Results
Fig. 4 shows the results of the spatial and memory ability test. In Fig. 4,
***13 <0.01
versus the control group, ##P <0.01 versus the group administered vehicle, "4T
<0.001
versus the group administered vehicle. $13 < 0.05, the group administered
aripiprazole
alone versus the group administered cilostazol and aripiprazole, P <0.05, the
group
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WO 2017/111123 PCT/JP2016/088554
administered cilostazol alone versus the group administered aripiprazole and
cilostazol.
[0173] The mice in the group administered vehicle spent less time within
the quadrant where
the platform was set during the training than the mice in the control group,
exhibiting
lowered spatial learning ability and memory ability. In contrast, the mice in
the group
administered cilostazol and aripiprazole spent significantly longer time
within the
quadrant than the mice in the group administered vehicle. In addition, the
mice in the
group administered cilostazol and aripiprazole spent significantly longer time
within
the quadrant than the mice in the group administered aripiprazole alone or
cilostazol
alone. The results reveal that the combinational effect demonstrated in
Example 1 is
also brought about in different test models.
